Terpene-quinones with antitumour activity

ABSTRACT

Terpene-quinone with antitumor activity defined from a cyclocondensation reaction of Diels-Alder, used to obtain families or series of said compounds having a new structure and presenting a cytotoxic activity to cellular cultures P-388, A-549, HT-29 and MEL-28, with levels of IC 50  lower than 1 microgram per unit of milliliter.

Present document relates to the developement of a series of novelproducts such as a new series of terpene-quinones, as well as theprocedure for its obtention.

Avarol and its quinone analogue, avarone are mero-sequiterpenoidsisolated from a sponge, Dysidea avara, which showed certain cytotoxicactivity and which can be considered as a model for a series ofanalogous, yet discovered or prepared afterwards, containing differentvariants of substitution at the quinone portion and/or the chain at thesesquiterpene portion. A good number of these compounds containing abicyclic serterpene system posess cytotoxic activity with values of IC₅₀arround 1 microgram per millilitre. Other sesquiterpene-quinones withmonocyclic terpene groups have also similar levels of activity.

Up to date, there has not been published any study on thestructure-activity relationship of these compounds regarding to size andtype of quinone and terpene groups of the molecule. Therefore, in thiswork it has been aimed to analyze the effect of the increase, diminutionand functionalization of terpene group and the expansion of quinonenucleus up to naphthoquinone.

In order to realize a clear exposition of the processes of theinvention, it will be attached to this text those reactions which aregoing to be explained hereinafter, written as their chemical formulae,said reactions resulting the invention's generic ones.

The molecules which are the objective or this work comprise a simple orsubstituted type A naphthoquinone as quinone system, in which R¹ is analkyl or alkoxy group and T matches an alkyl or cycloalkyl group,prefferably of isoprene, monoterpene, sesquiterpene type or with anintermediate size between these types. ##STR1##

Likewise, there are included as an objective those naphthoquinonederivatives of types B and C and the corresponding dihydroxynaphthaleneanalogous of types D and E.

Most of these compounds have been synthetized by using the Diels-Aldercyclocondensation, as a base-reaction, between conveniently substitutedp-benzoquinone and a compound comprising a conjugated diene system, inthe presence of a catalyst or not. The further Manipulation,equilibration or chemical modification of the directly resultingcycloaddition product (B) can successively yield the compounds of typesE, C, D and A which are represented below for two different kinds ofdiene system substitution.

This first kind of diene system substitution is shown in its reaction asfollows: ##STR2##

The second kind of diene system substitution fore mentioned will be:##STR3##

Starting compounds, useful for cycloadditions, have a conjugated dienesystem along carbon chains of the linear or branched alkane:2,6-dimethyloctane, 2,6,10-trimethyldodecane,2,6,10,14-tetramethylhexanedecane, 2,6,10,15,19,23-hexamethyltetracosaneor on any other of a cyclical nature biogenesically or artificiallyrelated to these.

Chemical names of those kinds of compounds refered in said genericreactions will be the following, taking into consideration that in saidnames there will appear numbers and words in brackets, meaning possiblepositions for a substituent and the possibility for said function orstructural modification to exist, respectively, and that they will beincluded in subtypes, depending on R¹ and T:

TYPE A COMPOUND

R¹ =Hydrogen 5(6)-alkyl(alkenyl)-naphtho-1,4-quinones5(6)-(poly)cycloalkyl(cycloalkenyl)-alkyl-naphtho-1,4-quinones 5(6)-(poly)cyclo(poly)isoprenyl-naphtho-1,4-quinones

R¹ =Alkyl 2(3)-alkyl-5(6)(7)(8)-alkyl(alkenyl)-naphtho-1,4-quinones2(3)-alkyl-5(6)(7)(8)-(poly)cycloalkyl(cycloalkenyl)-alkyl-naphtho-1,4-quinones2(3)-alkyl-5(6)(7)(8)-(poly)cyclo(poly)isoprenyl-naphtho-1,4-quinones

R¹ =Alkoxy 2(3)-alkoxy-5(6)(7)(8)-alkyl(alkenyl)-naphtho-1,4-quinones2(3)-alkoxy-5(6)(7)(8)-(poly)cycloalkyl(cycloalkenyl)-alkyl-naphtho-1,4-quinones2(3)-alkoxy-5(6)(7)(8)-(poly)cyclo(poly)isoprenyl-naphtho-1,4-quinones

R¹ =(di)Alkylamine2(3)-(di)Alkylamine-5(6)(7)(8)-alkyl(alkenyl)-naphtho-1,4-quinones 2(3)-(di)Alkylamine-5(6)(7)(8)-(poly)cycloalkyl(cycloalkenyl)-alkyl-naphtho-1,4-quinones2(3)-(di)Alkylamine-5(6)(7)(8)-(poly)cyclo(poly)isoprenyl-naphtho-1,4-quinones

TYPE B COMPOUND

R¹ =Hydrogen5(6)-alkyl(alkenyl)-4a,5,8,8a-tetrahydro-naphthalene-1,4-diones5(6)-(poly)cycloalkyl(cycloalkenyl)-alkyl-naphthalene-1,4-diones5(6)-(poly)cyclo(poly)isoprenyl-naphthalene-1,4-diones

R¹ =Alkyl 2(3) -alkyl-5(6)(7)(8)-alkyl(alkenyl)-4a,5,8,8a-tetrahydro-naphthalene-1,4-diones2(3)-alkyl-5(6)(7)(8)-(poly)cycloalkyl(cycloalkenyl)-alkyl-4a,5,8,8a-tetrahydro-naphthalene-1,4-diones2(3)-alkyl-5(6)(7)(8)-(poly)cyclo(poly)isoprenyl-4a,5,8,8a-tetrahydro-naphthalene-1,4-diones

R¹ =Alkoxy2(3)-alkoxy-5(6)(7)(8)-alkyl(alkenyl)-4a,5,8,8a-tetrahydro-naphthalene-1,4-diones2(3)-alkoxy-5(6)(7)(8)-(poly)cycloalkyl(cycloalkenyl)-alkyl-4a,5,8,8a-tetrahydro-naphthalene-1,4-diones2(3)-alkoxy-5(6)(7)(8)-(poly)cyclo(poly)isoprenyl-4a,5,8,8a-tetrahydro-naphthalene-1,4-diones

R¹ =(di)Alkylamine2(3)-(di)Alkylamine-5(6)(7)(8)-alkyl(alkenyl)-4a,5,8,8a-tetrahydro-naphthalene-1,4-diones2(3)-di)Alkylamine-5(6)(7)(8)-(poly)cycloalkyl(cycloalkenyl)-alkyl-4a,5,8,8a-tetrahydro-naphthalene-1,4-diones2(3)-(di)Alkylamine-5(6)(7)(8)-(poly)cyclo(poly)isoprenyl-4a,5,8,8a-tetrahydro-naphthalene-1,4-diones

TYPE C COMPOUND

R¹ =Hydrogen 5(6)-alkyl(alkenyl)-5,8-dihydro-naphtho-1,4-quinones5(6)-(poly)cycloalkyl(cycloalkenyl)-alkyl-5,8-dihydro-naphtho-1,4-quinones5(6)-(poly)cyclo(poly)isoprenyl-5,8-dihydro-naphtho-1,4-quinones

R¹ =Alkyl2(3)-alkyl-5(6)(7)(8)-alkyl(alkenyl)-5,8-dihydro-naphtho-1,4-quinones2(3)-alkyl-5(6)(7)(8)-(poly)cycloalkyl(cycloalkenyl)-alkyl-5,8-dihydro-naphtho-1,4-quinones2(3)-alkyl-5(6)(7)(8)-(poly)cyclo(poly)isoprenyl-5,8-dihydro-naphtho-1,4-quinones

R¹ =Alkoxy2(3)-alkoxy-5(6)(7)(8)-alkyl(alkenyl)-5,8-dihydro-naphtho-1,4-quinones2(3)-alkoxy-5(6)(7)(8)-(poly)cycloalkyl(cycloalkenyl)-alkyl-5,8-dihydro-naphtho-1,4-quinones2(3)-alkoxy-5(6)(7)(8)-(poly)cyclo(poly)isoprenyl-5,8-dihydro-naphtho-1,4-quinones

R¹ =(di)Alkylamine2(3)-(di)Alkylamine-5(6)(7)(8)-alkyl(alkenyl)-5,8-dihydro-naphtho-1,4-quinones2(3)-(di)Alkylamine-5(6)(7)(8)-(poly)cycloalkyl(cycloalkenyl)-alkyl-5,8-dihydro-naphtho-1,4-quinones2(3)-(di)Alkylamine-5(6)(7)(8)-(poly)cyclo(poly)isoprenyl-5,8-dihydro-naphtho-1,4-quinones

TYPE D COMPOUND

R¹ =Hydrogen 5(6)-alkyl(alkenyl)-naphthalene-1,4-diols and theirdiacetates(diesthers).5(6)-(poly)cycloalkyl(cycloalkenyl)-alkyl-naphthalene-1,4-diols andtheir diacetates(diesthers). 5(6)-(poly)cyclo(poly)isoprenyl-naphthalene-1,4-diols and theirdiacetates(diesthers).

R¹ =Alkyl 2(3)-alkyl-5(6)(7)(8)-alkyl-naphthalene-1,4-diols and theirdiacetates(diesthers).2(3)-alkyl-5(6)(7)(8)-(poly)cycloalkyl(cycloalkenyl)-alkyl-naphthalene-1,4-diolsand their diacetates(diesthers).2(3)-alkyl-5(6)(7)(8)-(poly)cyclo(poly)isoprenyl-naphthalene-1,4-diolsand their diacetates(diesthers).

R¹ =Alkoxy 2(3)-alkoxy-5(6)(7)(8)-alkyl(alkenyl)-naphthalene-1,4-diolsand their diacetates(diesthers).2(3)-alkoxy-5(6)(7)(8)-(poly)cycloalkyl(cycloalkenyl)-alkyl-naphthalene-1,4-diolsand their diacetates (diesthers).2(3)alkoxy-5(6)(7)(8)-(poly)cyclo(poly)isoprenyl-naphthalene-1,4-diolsand their diacetates(diesthers).

R¹ =(di)Alkylamine 2(3)-(di)Alkilamine-3(6)(7)(8)-alkyl(alkenyl)-naphthalene-1,4-diols and theirdiacetates(diesthers). 2(3)-(di)Alkilamine-5(6)(7)(8)-(poly)cycloalkyl(cycloalkenyl)-alkyl-naphthalene-1,4-diolsand their diacetates(diesthers).2(3)-(di)Alkilamine-5(6)(7)(8)-(poly)cyclo(poly)isoprenyl-naphthalene-1,4-diolsand their diacetates(diesthers).

TYPE E COMPOUND

R¹ =Hydrogen 5(6)-alkyl(alkenyl)-5,8-dihydro-naphthalene-1,4-diols andtheir diacetates(diesthers).5(6)-(poly)cycloalkyl(cycloalkenyl)-alkyl-5,8dihydro-naphthalene-1,4-diolsand their diacetates(diesthers).5(6)-(poly)cyclo(poly)isoprenyl-5,8-dihydro-naphthalene-1,4-diols andtheir diacetates(diesthers).

R¹ =Alkyl2(3)-alkyl-5(6)(7)(8)-alkyl(alkenil)-5,8-dihydro-naphthalene-1,4-diolsand their diacetates(diesthers).2(3)-alkyl-5(6)(7)(8)-(poly)cycloalkyl(cycloalkenyl)-alkyl-5,8-dihydro-naphthalene-1,4-diolsand their diacetates (diesthers).2(3)-alkyl-5(6)(7)(8)-(poly)cyclo(poly)isoprenyl-5,8-dihydro-naphthalene-1,4-diolsand their diacetates (diesthers).

R¹ =Alkoxy 2(3)-alkoxy-5(6)(7)(8)-alkyl(alkenyl)-5,8-dihydro-naphthalene-1,4-diols andtheir diacetates(diesthers).2(3)-alkoxy-5(6)(7)(8)-(poly)cycloalkyl(cycloalkenyl)-alkyl-5,8-dihydro-naphthalene-1,4-dialsand their diacetates(diesthers).2(3)-alkoxy-5(6)(7)(8)-(poly)cyclo(poly)isoprenyl-5,8-dihydro-naphthalene-1,4-diolsand their diacetates (diesthers).

R¹ =(di)Alkylamine2(3)-(di)Alkilamine-5(6)(7)(8)-alkyl(alkenyl)-5,8-dihydro-naphthalene-1,4-diolsand their diacetates(diesthers). 2(3)-(di)Alkilamine-5(6)(7)(8)-(poly)cycloalkyl(cycloalkenyl)-alkyl-5,8-dihydro-naphthalene-1,4-diolsand their diacetates(diesthers).2(3)-(di)Alkilamine-5(6)(7)(8)-(poly)cyclo(poly)isoprenyl-5,8-dihydro-naphthalene-1,4-diolsand their diacetates (diesthers).

With this list, types of compounds which result the objective of thework collected in this invention patent document become specified with ahigher precission, being fitted in types and subtypes according to thefore-mentioned explanation, formulae and reactions.

With said reactions and generic compounds, certain examples forpractical incorporations developed in the laboratory can be determined,the following rules having been taken in order to obtain the concretecompounds which will be hereinafter mentioned.

FIRST EXAMPLE

To a solution of 733 milligrams or 2.32 milliMole of methylmirceocomunate or compound 1, in anhydrous ether, another dry ethersolution of 251 milligrams or 2.32 milliMole of p-benzoquinone and acatalytic amount of BF₃ -etherate were added on stirring under inertatmosphere. Mixture was kept at room temperature for 24 hours. Water wasadded and it was washed up to neutralization. It was dried (Na₂ SO₄),filtered and distilled and a brown semisolid residue is obtained whichis subjectedto chromatography on silica-gel, so finally obtaining 127milligrams (13%) of 6-14-methoxy-14-oxo-drim-8(12)-en-11-il-methyl!-naphtho-1,4-quinoneorcompound number 2 and 560 milligrams (57%) of 5,8-dihydro-14-methoxy-14-oxo-drim-8(12)-en-11-il-methyl!-naphthalene-1,4-diol orcompound number 3. Acetylation of compound 3 under standard conditionsleads to an additional compound 3a named 5,8-dihydro-14-methoxy-14-oxo-drim-8(12)-en-11-il-methyl!-naphthalene-1,4-dioldiacetate. ##STR4##

Second Example

From 450 milligrams or 1.42 milliMole of methyl mirceocomunate orcompound 1, and 175 milligrams or 1.42 milliMole of2-methyl-p-benzoquinone, in thepresence of the same catalyst, underidentical conditions for 28 hours, andafter performing thechromatography, it was achieved 40 milligrams (6.5%) of the rearrangedproduct 4 or 2(3)-methyl-6-14-methoxy-14-oxo-drim-8(12)-en-11-il-methyl!-naphtho-1,4-quinone, ayellow oil, 170 milligrams of 5,8-dihydro-2(3)-methyl-6-14-methoxy-14-oxo-drim-8(12)-en-11-il-methyl!-naphtho-1,4-quinone orcompound 5, a reddish oil and further 298 milligrams (48%) of5,8-dihydro-2(3)-methyl-6-14-methoxy-14-oxo-drim-8(12)-en-11-il-methyl!-naphthalene-1,4-diol orcompound 6, a brown solid of melting point 97° C. Acetylation ofcompound 6, under standard conditions, leads to compound 6a or5,8-dihydro-2(3)-methyl-6-14-methoxy-14-oxo-drim-8(12)-en-11-il-methyl!-naphthalene-1,4-dioldiacetate. ##STR5##

Third Example

From 200 milligrams or 0.63 milliMole of methyl trans-comunate orreacting compound 7 and 68 milligrams or 0.63 milliMole ofp-benzoquinone, with thesame catalyst, conditions and operations than inthe first example, and after performing the chromatography, a highportion of the product having not reacted is recovered and it isobtained 95 milligrams (23%) of compound 8 or4a,5,8,8a-tetrahydro-6-methyl-5-14-methoxy-14-oxo-drim-8(12)-en-11-il-methyl!-naphthalene-1,4-quinone, ayellow oily product, and traces of compound9 which was acetylated up toyield compound 9a. ##STR6##

Fourth Example

From 1 gram or 7.35 milliMole of mircene or compound 10 in 150milliliters of anhydrous ether and 0.78 grams or 7.35 milliMole ofp-benzoquinone, in the presence of a small amount of BF₃ -etherate, thereaction being kept under the same fore conditions, for 36 hours andoperating as described in the first example, 1,65 grams of a reddishviscous residue are obtained, from which it is isolated, bychromatography on silica-gel, 150 milligrams (8.5%) of compound 11, or6-(4-methylpent-3-enil)-naphtho-1,4-quinone, a yellow-orange colouredoilyproduct, 300 milligrams (17%) of compound 12 or5,8-dihydro-6-(4-methylpent-3-enil )-naphtho-1,4-quinone with a similarconstitution and appearance and 825 milligrams (46%) of compound 13 or5,8-dihydro-6-(4-methylpent-3-enil)-naphthalene-1,4-diol, abrown-reddish solid of melting point 120° C. Acetylation of compound 13leads, under standard conditions, to compound 13a or5,8-dihydro-6-(4-methylpent-3-enil)-naphthalene-1,4-diol diacetate in ayield of 75% after purifying. Next, 198 milligrams or 0.6 milliMole ofcompound 13a will be dissolved into 50 milliliters of dichloromethane,and110 milligrams or 0.6 milliMole of metachloroperbenzoic acid (mcpba)and 150 milligrams or 2 milliMole of sodium hydrocarbonate are added.Mixture is kept under dry atmosphere and on stirring for 30 minutes.Reaction is finished by washing the organic phase with a solution of Na₂SO₄in water untill complete reduction of the excess oxidizing agent, itis subsequently washed with water up to neutralization, dried, filteredand distilled, so originating 153 milligrams of a colourless oilyresidue, which once subjected to chromatography, yields 120 milligrams(58%) of compound 14 or5,8-dihydro-6-(3,4-epoxy-4-nethylpentil)naphthalene-1,4-diol diacetate .##STR7##

Biological Activity

Cytotoxic activity of terpene-quinones obtained has been determined by asimple screening procedure using an adapted form from method describedby Bergeron et al. (1984) against cell cultures P-388 (suspensionculture of DBA/2 mouse lymphoid neoplasm), A-549 (single-layer cultureof human lung carcinoma), HT-19 (single-layer culture of human coloncarcinoma) and MEL-28 (single-layer culture of human melanoma). Thevalues of IC₅₀ in microM., observed for representative compoundsmentioned on this work, are listed in the following table, in whichthere are also included the values found for avarone, as an element forcomparison.

    ______________________________________                                        Compound   P-388  A-549      HT-29 MEL-28                                     ______________________________________                                        Avarone    3.1    6          6     6                                           2         0.12   0.23       0.23  0.23                                        3         0.23   0.28       0.28  0.28                                        3a        0.24   0.24       0.5   0.5                                         3b        0.2    0.2        1     0.2                                         3c        0.3    1.1        1.1   0.6                                         4         1.1    1.1        2.3   2.3                                         5         0.58   1.2        2.3   1.2                                         8         1.2    2.3        2.3   2.3                                        12         1.0    2.1        2.1   2.1                                        13         2.1    5.9        5.9   5.9                                        14         1.4    7.0        7.0   7.0                                        ______________________________________                                    

P-388 cells are sown into 16 millimeters wells at 1×10,000 cells perwell in alliquots of 1 millilitre of MEM 5FCS containing the indicateddrug concentration. Separately, a batch of drug-less cultures is sown asacontrol for growth to assure that cells are kept at a logarithmicgrowth stage. All determinations are made in duplicate. After three daysof incubation at 37° C., 10% CO₂ in a 98% humidity atmosphere,approximate IC₅₀ is determined by comparing growth on drug-wellswithgrowth on control-wells.

A-549, HT-29 and MEL-28 calls are sown into 16 millimeters wells at2×10,000 cells per well in alliquots of 1 millilitre of MEM 10FCScontaining the indicated drug concentration. Separately, a batch ofdrug-less cultures is sown as a control for growth to assure that callsare kept at a logarithmic growth stage. All determinations are made induplicate. After three days of incubation at 37° C., 10% CO₂ in a 98%humidity atmosphere, wells are stained with 0.1% Crystal Violet.Approximate IC₅₀ is determined by comparing growth on drug-wellswithgrowth on control-wells.

Once the nature of invention procedure and the obtention of productspointed out in the present report are sufficiently described, it onlyremains for us to add that said procedure may be partially varied,withoutinvolving any substantial alteration of characteristics claimedhereinafter.

We claim:
 1. A compound of the formula: ##STR8## wherein R¹ is selectedfrom the group consisting of hydrogen, alkyl, alkoxy, alkylamine anddialkylamine; and wherein and T is either an alkyl or a cycloalkyl groupcontaining one or more isoprene units.
 2. A compound of claim 1, whereinT comprises an isoprene, monoterpene, or sesquiterpene group.
 3. Acompound of the formula: ##STR9## wherein R¹ is selected from the groupconsisting of hydrogen, alkyl, alkoxy, alkylamine and dialkylamine; andwherein and T is either an alkyl or a cycloalkyl group containing one ormore isoprene units.
 4. A compound of claim 3, wherein T comprises anisoprene, monoterpene, or sesquiterpene group.
 5. A compound of theformula: ##STR10## wherein R¹ is selected from the group consisting ofhydrogen, alkyl, alkoxy, alkylamine and dialkylamine; and wherein and Tis either an alkyl or a cycloalkyl group containing one or more isopreneunits.
 6. A compound of claim 5, wherein T comprises an isoprene,monoterpene, or sesquiterpene group.
 7. A compound of the formula:##STR11## wherein R¹ and R² are each independently selected from thegroup consisting of hydrogen, alkyl, alkoxy, alkylamine anddialkylamine; and wherein and T is either an alkyl or a cycloalkyl groupcontaining one or more isoprene units.
 8. A compound of claim 7, whereinT comprises an isoprene, monoterpene, or sesquiterpene group.
 9. Acompound of the formula: ##STR12## wherein R¹ and R² are eachindependently selected from the group consisting of hydrogen, alkyl,alkoxy, alkylamine and dialkylamine; and wherein and T is either analkyl or a cycloalkyl group containing one or more isoprene units.
 10. Acompound of claim 9, wherein T comprises an isoprene, monoterpene, orsesquiterpene group.
 11. The compound 6-14-methoxy-14-oxo-drim-8(12)-en-11-il-methyl!-naphtho-1,4-quinone. 12.The compound 5,8-dihydro-6- 14-methoxy-14-oxo-drim-8(12)-en-11-il-methyl!-naphthalele-1,4-diol.
 13. Thecompound 5,8-dihydro-6-14-methoxy-14-oxo-drim-8(12)-en-11-il-methyl!-naphthalele-1,4-dioldiacetate.
 14. The compound 2(3)-methyl-6-14-methoxy-14-oxo-drim-8(12)-en-11-il-methyl!-naphtho-1,4-quinone. 15.The compound 5,8,-dihydro-2(3)-methyl-6-14-methoxy-14-oxo-drim-8(12)-en-11-il-methyl!-naphtho-1,4-quinone. 16.The compound 5,8-dihydro-2(3)-methyl-6-14-methoxy-14-oxo-drim-8(12)-en-il-methyl!-naphthalene-1,4-diol.
 17. Thecompound 5,8-dihydro-2(3)-methyl-6-14-methoxy-14-oxo-drim-8(12)-en-il-methyl!-naphthalene-1,4-dioldiacetate.
 18. The compound 4a,5,8,8a-tetrahydro-6-methyl-5-14-methoxy-14-oxo-drim-8(12)-en-il-methyl!-naphthalene-1,4-quinone. 19.The compound 6-(4-methylpent-3-enil)-naphtho-1,4-quinone.
 20. Thecompound 5,8-dihydro-6-(4-methylpent-3-enil)-naphtho-1,4-quinone. 21.The compound 5,8-dihydro-6-(4-methylpent-3-enil)-naphthalene-1,4-diol.22. The compound5,8-dihydro-6-(4-methylpent-3-enil)-naphthalene-1,4-diol diacetate. 23.The compound5,8-dihydro-6-(3,4-epoxy-4-methylpentil)-naphthalene-1,4-diol diacetate.24. The compound 6- 14-methoxy-14-oxo-drim-8,(12)-epoxi-11-yl-methyl!-naphthalene-1,4-diol diacetate.
 25. 6-14-methoxy-14-oxo-drim-8(12)-en-11-yl-methyl!-naphthalene-1,4-dioldiacetate.
 26. 5,8-dihydro-6-epoxi-6- 14-oxo-drim-8-oxo-11-yl-methyl!-naphthalene-1,4-diol.
 27. 2(3)-methyl-5-14-methoxy-14-oxo-drim-8(12)-en-yl-methyl!-naphtho-1,4-quinone.